AIMS:Little is known about the endogenous function of the CYP2Cs. We postulate that these enzymes have important roles in the metabolism of physiologically important endogenous substrates such as arachidonic acid and retinoic acid (Vitamin A). Our goal has been to identify all CYP2Cs in the mouse, determine their contribution to the production of particular eicosanoid metabolites and to identify their physiological role in these tissues. The P450-derived AA metabolites are known to possess potent biological actions in numerous tissues. These effects depend on the regio- and stereochemistry of the products. We are attempting to determine the organ and cell-specific localization of these isoforms. A long-range goal is to produce knockout mice to study the physiological importance of this class of enzymes, and to overexpress these enzymes in cell lines to further establish their function.ACCOMPLISHMENTS These studies were initiated to determine the endogenous function of the CYP2Cs using the mouse as a model. Our laboratory recently cloned five members of the mouse CYP2C subfamily (CYP2C29, CYP2C37, CYP2C38, CYP2C39 and CYP2C40), expressed the recombinant P450 proteins in E. coli, and showed that they are active in the metabolism of arachidonic acid. We determined the organ and cell-specific localization of these isoforms using Western blots, PCR and cloning techniques and immunohistochemistry. Western blotting indicated and RT-PCR indicted that lung contained relatively large amounts of CYP2C29, . CYP2C40 was found in relatively high amounts in colon, cecum, gut, in kidney and heart. CYP2C40 metabolized arachidonic acid (AA) to 16R- and 16S-HETE . This is the first P450 found to produce 16-HETE as a primary product. Intestinal microsomes also produced 16-HETE. 16R-HETE is thought to be important in processes such as renal vasodilation and inhibiting neutrophil aggregation and adhesion. Using universal primers and subcloning and sequencing the products, the 2C mRNAs in colon and cecum were identified as solely CYP2C40, kidney contained CYP2C40 and a new unidentified member of the CYP2Cs. Lung contained mainly CYP2C29 and some CYP2C40. CYP2C29 was found in endothelial cells lining blood vessels, with some CYP2C40. Western blotting indicated and RT-PCR indicted that lung contained large amounts of CYP2C29. Endothelial cells of arteries contained CYP2C29 and some CYP2C40.These studies indicate that CYP2Cs may be important physiological enzymes in heart, gut, colon, endothelial cells, and lung.